A novel two EF-hand Ca2+-binding allergen from ragweed pollen

Abstract

Ragweed pollen represents the major source of allergenic proteins in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is not a major cause of pollinosis, but it is rapidly increasing. Here we reported the isolation of cDNAs coding for a pollen-specific two EF-hand calcium binding allergen from ragweed pollen showing homology to the calcium binding allergens from Bet v 4, Phl p 7, and other polcalcins. Calcium binding proteins (CBP) contain a variable number of EF-hand sequence motifs, which consist of an a-helix, a loop around the calcium ion and a second a-helix. Furthermore, CBPs were also described as relevant cross-reactive pollen allergens. Twenty-two cDNA clones (coding for nine isoforms) have been identified by IgE immunoscreening as CBPs containing two EF-hand domains. Two of nine isoforms were expressed in E.coli as soluble his tagged fusion proteins and purified to homogeneity. Immunoblots using rabbit anti-Phl p 7 antiserum demonstrated no cross-reactivity of the Phl p 7 antiserum with the Bet v 4 homologues in ragweed, although the sequence homology between these proteins was well above 50%. Testing various calcium-binding proteins with a large panel of pollen allergic patients primary sensitized to grass and tree pollen we found that most of the patients recognized Phl p 7 and Bet v 4. Only a small percentage displayed IgE antibodies reacting with ragweed CBPs. The observed limited cross-reactivity is probably explained by the fact that sequence homology among CBPs is extremely variable and mostly confined to the EF-hand domains.